The invention relates to a method and an apparatus for executing the method and separation media for separating particles in a free-flow electrophoresis apparatus, in particular for separating cells or complexes thereof, cellular constituents, organelles or biomolecules and of parts or complexes thereof in accordance with the generic term indicated in the claims.
A major difficulty in diagnostic practice and research involves precisely defined sample preparations. However, the technologies in common use today, such as centrifugation, filtration, magnetic separation and FACS (Fluorescence Activated Cell Sorting) have various disadvantages, such as restricted specificity, low throughput rates, limited purification ability or also expensive apparatus for isolating and purifying cells, organelles or biomolecules (e.g. protein complexes). The most important applications for purifying cells, organelles and biomolecules (e.g. protein complexes) are found in the fields of Biopharmacy, Biotechnology, Environmental Analysis, Food Technology and Medicine. The applied techniques are either preparative (Biopharmacy and Biotechnology) or analytic (Environmental Analysis, Food Technology and Medicine) in scope.
Free-flow electrophoresis (FFE) is one of the most promising technologies for separating all possible particles [cf. Krivanova L. & Bocek P. (1998), “Continuous free-flow electrophoresis”, Electrophoresis 19: 1064–1074]. In the field of Proteomics FFE is the technology of choice for the defined preseparation of complex protein samples in terms of their varying pI values (degree of ionization). Using FFE, the cells are separated on the basis of the electrophoretic mobility of the cells. The corresponding principles have already been characterized in depth [cf. e.g. Bondy B., Bauer J., Seuffert I. and Weber G. (1995), “Sodium chloride in separation medium enhances cell compatibility of free-flow electrophoresis”, Electrophoresis 16: 92–97], whereas FFE has received little recognition as most cell types differ only minimally in terms of their surface charge, making the separation of these cell types difficult. One of the first refinements was the introduction of immuno-FFE which allowed specific antibodies to be bound to specific surface epitopes of the cells to be separated, making it possible to modify the electrophoretic mobility of these cells in FFE by modifying the net charge of the cell surface [cf. e.g. Hansen E. and Hannig K. (1982), “Antigen-specific electrophoretic cell separation (ASECS): Isolation of human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies”, J. Immunol. Methods 11, 51: 197–208].
An FFE method of the same generic type is described in the international patent application PCT/EP01/14408. It concerns an electrophoresis method employing an apparatus with a separation chamber through which the separation medium flows and which is a space defined by a floor and a cover and spacers separating these two from each other. In addition, this FFE apparatus encompasses a pump for supplying the separation medium which enters the separation chamber via medium feed lines and leaves the chamber via outlets. The FFE apparatus also includes electrodes for applying an electric field within the separation medium and sample injection points for adding the mixture of particles or analytes and fractionation points for removing the particles separated by FFE in the separation medium. These separated particles can be used for analytic purposes or for further preparative processing. In order to exert an influence upon the separation medium flow profile provision can be made for two or more separate dosage pump feed lines to add medium which are connected to the separation chamber in the vicinity of the fractionation points in the vicinity of the electrodes.
The patent U.S. Pat. No. 5,948,231 discloses compositions, methods, and apparatus for performing ultrafast binding assays by capillary electrophoresis or other electro-separation techniques, such as Free Flow Electrophoresis.
The aim of the present invention is to refine the preparative and analytic isolation of particles such as cells, organelles and biomolecules (e.g. protein complexes) and similar matter and that of bioparticles or biopolymers in FEE.
In accordance with the invention this aim is fulfilled in accordance with a first aspect by the proposal of a free-flow electrophoresis method for separating particles, in particular for separating cells, cell constituents, organelles or biomolecules and parts or complexes thereof.
In accordance with the invention this aim is fulfilled in accordance with a second aspect by the proposal of a free-flow electrophoresis apparatus for executing the method for separating particles, in particular for separating cells, cell constituents, organelles or biomolecules and parts or complexes thereof.
In accordance with the invention this aim is fulfilled in accordance with a third aspect by the proposal of separation media for separating particles, in particular for separating cells, cell constituents, organelles or biomolecules and parts or complexes thereof in a free-flow electrophoresis apparatus.
Additional preferred characteristics are indicated in each of the dependent claims.
In the context of the present invention, a particle includes for instance all particle units of mass preferably of biological origin, such as cells, viruses, cell organelles, vesicles, cell nuclei, membranes and parts or aggregates thereof; in addition, biomolecules such as lipids, proteins, DNA, RNA and glucose and complexes or aggregates of biomolecules such as lipoproteins, glycoproteins, lipo-polysaccharides etc. These particles also include organic and inorganic molecules such as pharmaceuticals, polymers and similar substances.
A method in accordance with the invention for separating such particles on the basis of selectively modified surface charges, in particular for separating cells, cell constituents, organelles or biomolecules and parts or complexes thereof using free-flow electrophoresis techniques, encompasses the following advantages:                Magnetic beads are not suitable for the preparative separation of particles; moreover, only a single type of particle can be isolated at one time using magnetic beads. Magnetic bead applications can therefore be substituted by the use of FFE in accordance with the invention—if positively and negatively charged labels are employed simultaneously. The FFE in accordance with the invention allows the simultaneous isolation of at least two types of particles with maximum efficiency as the individual electrophoretic mobility of the particles is increased considerably and selectively.        Many FACS applications can be substituted by FFE applications and executed by facilities which are considerably cheaper and faster and, if required, automated.        Specifically selected, living cells can be qualitatively and quantitatively isolated and isolated on a preparative or analytic scale thanks to the specificity of the antibodies used and the separation force of FEE.        For the first time it is possible to prepare samples gently for extracting a highly-purified population of organelles.        Particle mixtures can be accurately separated on a preparative and analytic scale in relatively large quantities within a short period. One example is provided by the purification of circulating tumor cells from whole blood (tumor cells are found in whole blood at the ratio of 1:106 to 1:108): the white blood cells (2 ml MNC fraction from 10 ml whole blood), including the tumor cells, were separated from the red blood cells in whole blood treated with heparin using a density gradient and after appropriate marking of the tumor cells (incubation with epitope-specific antibodies with charge labels) were subjected to FEE for separation. They were added at a rate of 4–10 ml per hour. The purified tumor cells were able to be extracted in 2–8 ml volumes.        The proposed free-flow electrophoresis (FFE) apparatus allows various separation techniques to be conducted using a range of selective separation parameters.        The proposed FFE method allows not only the isolation but also the selective depletion of particles from a particle mixture, such as for instance, the depletion of abundant proteins (e.g. separating serum albumin from bodily fluids, for instance blood plasma).        The adhesion of particles to the electrodes that are separated due to their particular biological relevance and the subsequent major loss of these particles, can be successfully prevented by the use of a focusing buffer consisting of an electrically highly-conductive buffer medium.        
Preferred and exemplary embodiments of the method in accordance with the invention and of the apparatus in accordance with the invention for separating particles on the basis of their selectively modified net surface charge (especially for separating cells or cell constituents, organelles or biomolecules and parts or complexes thereof using free-flow electrophoresis method) shall be described in more detail below on the basis of schematic drawings which serve only to illustrate the invention and in no way restrict its scope. In the drawing: